抗头孢氨苄单克隆抗体的制备与初步应用

doi:10.16431/j.cnki.1671-7236.2017.04.031

Abstract

Abstract:

The aim of the study was to prepare a monoclonal antibody (McAb) against cephalexin(CEX),and preliminarily establish an ELISA method to detect residues of CEX in milk. The CEX was conjugated to carrier bovine serum albumin (BSA) and ovalbumin (OVA) by two-step glutaraldehyde method. BALB/c mice were immunized with the prepared CEX-BSA conjugate. After five times immunization, we built and screened two strains of hybridoma cells (2G4 and 5B3) which secreted McAb against CEX by hybridoma technology.The 2G4 cell lines were injected into BALB/c mouse's abdominal cavity to produce ascites. Finally the ascites were purified and identified. The results showed that antibody titer of the McAb 2G4 was above 1∶1.28×10⁵,its antibody subtype was IgG2b(κ) and affinity constant K was 1.51×10⁹ L/mol. The cross experiment result evidenced that the cross reaction rate of cefradine was 52.55%, ceftiofur, ceftriaxone, cefotaxime, cephalothin, penicillin, streptomycin and tetracycline had no cross reaction. We established an ELISA method to detect CEX residues in milk and its linear range was 20 to 1 000 ng/mL, linear equation y=0.4674x-0.5359(R²=0.9909), its limit of detection (LOD) was 19.68 ng/mL and the average recovery rate was 91.31%, the average coefficient of variation was below 15%. The detection limit was lower than the maximum residue limit of cefalexin in China and European Union. So this detection method might have good application foreground.

Key words: monoclonal antibody; cephalexin; ELISA; artificial antigen

CLC Number:

S859.79⁺6

ZHANG Wei, WANG Xing, LIU Jin-ping, XU Sheng-le, QIN Hai-yan, LIU Ruo-xuan, CAI Yun-hong, RU Xiao-fei, HOU Ke, WANG Han-dong. Preparation and Preliminary Application of Anti-cephalexin Monoclonal Antibody[J]. , 2017, 44(4): 1159-1167.

References

[1] 张伟,王兴,刘金萍,等. 动物性食品中头孢氨苄残留检测研究进展[J].安徽农业科学,2016,44(19):84-86.
[2] Kelkar P S,Li J T. Cephalosporin allergy[J].New England Journal of Medicine,2001,345(11):804-809.
[3] Singla A. Uncommon cause of acute drug-induced liver injury following mammoplasty[J].Gastroenterology Research,2010,56(4):171-172.
[4] Park H J,Oh J H,Hwang J Y,et al. Identification of gene-based potential biomarkers for cephalexin-induced nephrotoxicity in mice[J].Molecular and Cellular Toxicology,2006,2(3):193-201.
[5] Sanjana R K,Shah R,Chaudhary N,et al. Prevalence and antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) in CMS-teaching hospital:A preliminary report[J].Journal of College of Medical Sciences-Nepal,2010,6(1):141-146.
[6] 农业部畜牧兽医局. 农业部发布动物性食品中兽药最高残留限量(续)[J].中国兽药杂志,2003,37(3):5-11.
[7] Prasad G V,Sravani S,Ishaq B M,et al. Development and validation of UV-spectrophotometric method for determination of cephalexin[J].Asian Journal of Research in Chemistry,2013,6(5):490-494.
[8] Oliveira R V,De Pietro A C,Cass Q B. Quantification of cephalexin as residue levels in bovine milk by high-performance liquid chromatography with on-line sample cleanup[J].Talanta,2007,71(3):1233-1238.
[9] 倪梅林,崔晓美,殷居易,等. 动物组织与水产品中头孢氨苄残留量的液相色谱-串联质谱检测[J].分析测试学报,2009,28(4):489-492.
[10] 汪雪雁,祁克宗,陈玎玎,等. 鸡肉中头孢类抗生素的MISPE-HPCE检测[J].江苏农业学报,2012,28(1):193-197.
[11] Chen L,Wang Z,Ferreri M,et al. Cephalexin residue detection in milk and beef by ELISA and colloidal gold based one-step strip assay[J].Journal of Agricultural & Food Chemistry,2009,57(11):4674-4679.
[12] 卞素敏,扈洪波,邢仕歌,等. ELISA法检测牛奶中头孢氨苄残留[J].食品科学,2013,34(20):186-189.
[13] Zhang J,Wang Z,Mi T,et al. A homogeneous fluorescence polarization immunoassay for the determination of cephalexin and cefadroxil in milk[J].Food Analytical Methods,2014,7(4):879-886.
[14] Girolamo F D I,Ponzi M M,Alessandroni J,et al. A simple and effective method to analyze membrane proteins by SDS-PAGE and MALDI mass spectrometry[J].Anticancer Research,2010,30(4):1121-1129.
[15] 刘秀梵. 单克隆抗体在农业上的应用[M]. 合肥:安徽科学技术出版社,1994.
[16] Beatty J D,Beatty B G,Vlahos W G. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay[J].Journal of Immunological Methods,1987,100(1-2):173-179.
[17] 胡文丽,匡雪,简宗辉,等. 抗STa多克隆抗体的制备、鉴定和纯化[J].中国畜牧兽医,2015,42(5):1082-1087.
[18] 王照鹏,李向梅,徐飞,等. 大观霉素人工抗原的合成及其多克隆抗体制备[J].中国畜牧兽医,2014,41(2):54-57.
[19] 刘成梅,黄丽,罗舜菁,等. 氨苄青霉素人工抗原的制备及鉴定[J].食品科学,2007,28(9):384-388.
[20] 赵启法. 头孢氨苄人工抗原的合成及单克隆抗体的制备[D]. 郑州:郑州大学,2012.
[21] 彭会建,王捍东,王宗元,等. SMD单克隆抗体的制备及测定方法的建立[J].畜牧与兽医,2003,35(9):12-14.
[22] 马泓冰. 几种单克隆抗体纯化方法的比较性研究[D]. 苏州:苏州大学,2005.
[23] 唐佳佳,李小兵,刘国文,等. 单克隆抗体纯化的研究进展[J].中国畜牧兽医,2011,38(2):76-80.

Preparation and Preliminary Application of Anti-cephalexin Monoclonal Antibody

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[2]	MU Yijiao, YU Ruiming, ZHANG Liping, WANG Yonglu. Establishment and Application of ELISA for Detection of IgA Antibody Based on the Recombinant Full-length S2 Protein of Porcine Epidemic Diarrhea Virus Variant Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1185-1194.
[3]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[4]	HOU Haoyu, ZHENG Nannan, WU Hongju, CHEN Yinlong, LI Chao, ZHANG Angke, JI Pengchao, WAN Bo, DU Yongkun, ZHANG Gaiping. Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1241-1249.
[5]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[6]	HAN Rui, YANG Xing, ZHANG Wenbo, CHEN Jialing, ZHOU Xiaoli. Prokaryotic Expression of Autolysin from Streptococcus suis and Establishment of an Indirect ELISA Method for Detection of Its Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3508-3519.
[7]	TANG Qingfeng, LI Ling, HUANG Jiaxiang, FANG Wenyuan, NONG Haoru, TANG Yan, LU Chengwei, WEI Panqiu, FENG Ling. Survey on Mycotoxin Contamination of Buffalo Milk in Different Seasons Under Different Feeding Modes in Guangxi [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3665-3675.
[8]	ZHANG Wenyan, WANG Yawen, YUAN Chen, FENG Yawen, TENG Zhaojian, SHANG Jialiang, LU Jicheng, SONG Qinye. Establishment of Indirect ELISA Method for Detection of African Swine Fever Virus Antibodies Based on Truncated p72 Protein [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(7): 2688-2697.
[9]	TIAN Guangyuan, WANG Yuchen, ZHOU Yaping, GUO Ting, ZHAO Hongmei, ZHAO Fengmiao, SUN Yajie, BIAN Yuchen, YU Jialiang, ZHOU Yuxia. Prokaryotic Expression and Indirect ELISA Method Establishment of P60_58aa-928aa Protein of Mycoplasma ovipneumoniae [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1960-1969.
[10]	CHEN Yan, XUE Qiang, JIN Yong, ZOU Mingqiang, QI Xiaohua, LI Boyi, YIN Hong. Devolopment of Surface-enhanced Raman Spectroscopy Combined with Immunochromatography for Detecting Cephalexin [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1577-1584.
[11]	WANG Leming, WANG Yurui, ZENG Zixuan, RAO Guoshun, WU Zhengjiao, JIN Weikun, WANG Dongying. Preparation of Cathepsin L1 Monoclonal Antibody Against Fasclala gigantica and Establishment of Double Antibody Sandwich ELISA [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 677-686.
[12]	MO Hongfang, JIE Kai, CHEN Xin, WEN Wei, LIU Wenqiang, CHEN Huanchun, QIAN Ping, LI Xiangmin. Preparation and Identification of Monoclonal Antibodies Against Porcine Seneca Valley Virus [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 307-317.
[13]	CHEN Jing, WU Yan, LI Mei, YUE Jun, ZHU Erpeng, WEN Ming, ZHANG Shuangxiang, CHENG Zhentao. Prokaryotic Expression of LPPA Protein of Mycoplasma mycoides subsp. capri and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 318-327.
[14]	HU Huanyi, WANG Jianzhong, LIU Changjin, LIN Min, LIU Xiaolan, WEI Huangsiwu, DENG Shunzhou. Eukaryotic Expression of Green Fluorescent Protein and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 328-337.
[15]	YANG Yuxin, ZHAO Fuhua, LIU Yu, QIU Jicheng, CAO Yuying, ZHANG Lu, BAI Runian, WANG Jianzhong, CAO Xingyuan. Pharmacokinetics and Bioequivalence of Two Cephalexin Formulations in Beagle Dogs [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(1): 386-394.